Dendritic cell-specific antibodies

ABSTRACT

Isolated antibody or preparation of antibodies comprising an antigen-binding domain wherein the antigen is present on activated dendritic cells and wherein the antibody does not interact with CMRF-44 antigen or CD83 antigen.

The present application is a 371 of PCT/NZ97/00134, filed Oct. 9, 1997,and claims priority from New Zealand patent application Serial No.299537, filed Oct. 9, 1996.

FIELD OF THE INVENTION

This invention relates generally to immunological reagents (antibodies)capable of binding to activated dendritic cells, to cell lines whichexpress such antibodies and to a process for identifying and purifyingdendritic cells from blood using such antibodies.

BACKGROUND OF THE INVENTION

Dendritic cells (DC) constitute a distinct group of potent antigenpresenting cells (APC) which are bone marrow derived and found as tracepopulations in the circulation as well as within both lymphoid andnonlymphoid tissues¹⁻³. Although their importance as the most effectivehaemopoletic cell involved in the initiation of primary immune responseshas been well demonstrated⁴⁻⁷, no human DC specific lineage marker hasbeen identified and most features of their ontogeny and relationship toother leukocytes remains unclear.

Phenotypically, human DC are characterised^(1-3.7-11) by a high densityof class II MHC antigens, the presence of a wide range of adhesionmolecules and the absence or low expression of a range of lineagespecific cell surface antigens (CD3, CD14, CD16, CD 19, CD57). A numberof activation antigens including IRAC¹², HB15¹³, 4F2⁸, IL-2R^(7.8), andB7/BB-1^(7.14) have also been reported on human DC, particularly afteractivation, although the anti-IRAC and HB15 reagents have not been shownto stain isolated fresh blood DC. Despite this phenotypiccharacterisation, identification and therefore purification of DCremains difficult as the majority of these antigens are expressed byother resting and activated cell types. Many of the functional andphenotypic features of DC are shared by both Hodgkins cells (HC) andHodgkins Disease (HD) derived cell lines and there is increasingevidence to support the hypothesis, that in some instances, HC representa malignant form of DC¹⁵⁻¹⁷.

Immunological reagents for use in a process for identifying andpurifying DC therefore have obvious utility. Such reagents will need torecognize epitopes or antigens specific to DC. To date antibodies havebeen generated against early activation antigens CD83^(18.19) andCMRF-44²⁰. However, there remains a need to have available antibodieswhich can bind to different epitopes on DC than CD83 and CMRF-44.

It is therefore an object of this invention to provide immunologicalreagents which recognise epitope(s) of a novel activation antigen foundon DC or at least provide the public with a useful choice.

SUMMARY OF THE INVENTION

In a first aspect the present invention can be said to provideantibodies or binding fragments thereof which specifically bind to DCactivation antigen CMRF-56.

Conveniently, the antibody is a monoclonal antibody, preferablymonoclonal antibody CMRF-56 (mAb CMRF-56).

In yet a further aspect, the invention provides hybridoma cell lineCMRF-56.

In still a further aspect, the present invention provides mAb CMRF-56secreted by hybridoma cell line CMRF-56 which specifically binds to anepitope on activated human DC but does not bind to activation antigensCMRF-44 and CD83.

In yet a further aspect, the invention provides an antibody or antibodybinding fragment which is specific for the epitope on human DC to whichmAb CMRF-56 binds.

In still a further aspect, the present invention provides a process foridentifying activated DC in a sample containing such cells comprisingthe step of contacting said sample with an antibody or antibody bindingfragment as defined above.

In yet a further aspect, the invention provides a process for purifyingand/or concentrating DC from a sample containing such cells comprisingthe step of contacting said sample with an antibody or antibody bindingfragment as defined above.

In the preferred embodiment of these processes, the cells to beidentified or purified are activated human DC and the antibody is mAbCMRF-56.

In still a further aspect, the invention provides a DC purificationsystem for use in purifying or concentrating DC from a sample containingsuch cells which includes an antibody or antibody binding fragment asdefined above.

Conveniently, the purification system is designed to purify activatedhuman DC and the antibody is optionally labelled mAb CMRF-56.

In still a further aspect, the present invention consists in activatedDC recovered by a process as defined above or by using a purificationsystem as defined above.

In yet a further aspect, the invention provides an immunopotentiatingcomposition comprising activated DC obtained as above and at least oneantigen capable of generating a protective immunological response to adisease in an animal susceptible to such disease.

In still a further aspect, the invention provides an immunopotentiatingcomposition comprising an antibody as defined above and at least oneantigen capable of generating a protective immunological response to adisease in a patient susceptible to such disease.

In still a further aspect, the invention provides an immunopotentiatingcomposition comprising activated DC obtained as above, an antibody asdefined above and at least one antigen capable of generating aprotective immunological response to a disease in a patient susceptibleto such disease.

In still a further aspect, the invention provides an immunopotentiatingcomposition comprising an antibody as defined above.

In still a further embodiment, the invention provides a method ofprophylaxis and/or therapy in relation to a disease which comprisesadministering to a subject susceptible to said disease animmunopotentiating composition as defined above.

In yet a further aspect, the invention provides an assay kit whichincludes mAb CMRF-56 for use as a diagnostic marker of activated DC.

SUMMARY OF THE DRAWINGS

While the present invention is broadly as defined above, it will beappreciated that it is not limited thereto but that it also includesembodiments of which the following description provides examples. Inaddition, the present invention will be better understood by referenceto the accompanying drawings which are as follows:-

FIG. 1 shows the reactivity of mAb CMRF-56 with blood and tonsilleukocytes. (A) (i) Fluorescent intensity histogram of granulocytesstained with mAb CMRF56(filled) or isotype control (ii) dot plots ofER⁻PBMC double labelled with CMRF-56 vs CD3, CD14, CD16, CD19-PE (iii)dot plot of ER⁺PBMC double labelled with CMRF56 vs CD3-PE. (B) Dot plotsof cultured (16 h. 37° C.) ER⁻PBMC double labelled with CMRF-56, CD83 orCMRF-44 vs CD19-PE. (C) Dot plots of tonsil ER⁻ lymphocytes doublelabelled with CMRF-56, CD83 or CMRF-56 vs CD19-PE. In all cases thegates delineating positive staining shown were set on the basis ofnegative control staining. Data are from representative experiments.

FIG. 2 shows the reactivity of CMRF-56 and HB15 (CD83) with human celllines and CD83 Cos cell transfectants (A) Data for the human cell linesL428 and Jurkat are shown as immunofluorescent profiles obtainedfollowing labelling with either isotype controls ( - - - ), CMRF-56 orCD83 ( - - - ) and are from a representative experiment of sixperformed. The intensity of CMRF56 and HB15 labelling (MFI) over that ofthe negative controls is shown in the right hand corner of eachhistogram of the human cell lines. (B) Data for the COS celltransfectants are shown as the immunofluorescent profiles obtainedfollowing labelling of either control transfectants ( - - ) or CD83transfectants ( - - - ) with CMRF-56 or CD83. Data are from arepresentative experiment of three performed.

FIG. 3 characterises the CMRF-56 antigen. Binding of CMRF-56, CD83 andnegative control mAb to human Ig, CD83-Ig and CD83-histidine as analysedby ELISA. Data are shown as histograms and are from a representativeexperiment of three performed.

FIG. 4 shows expression of CMRF-56 on directly isolated DC. (A) Directlyisolated blood DC were cultured in medium for 0, 3,6 or 12 h thenanalysed by double labelling with CMRF-56, CMRF-44 or CD83 vs DR-PE. Inall cases the gates delineating positive staining shown were set on thebasis of negative control staining. Data are from a representativeexperiment of 3 performed.

FIG. 5 shows an analysis of CMRF-56 expression within cultured lowdensity preparations of ER⁻BMC (A) Dot plots of preparations doublelabelled with CMRF-44, CD83 or CMRF-56 vs a mix of PE conjugated CD3,CD14, CD16 and CD19 mAb (B) Dot plots of preparations double labelledwith CD83 or CMRF-44 vs CMRF-56 biotin. in all cases the gatesdelineating positive staining shown were set on the basis of negativecontrol staining. (C) Allogeneic MLR performed following sorting of alow density preparation on the basis of CMRF-56 expression. The CMRF-56positive (Δ) and negative (∇) populations together with unlabelled (⋄)and labelled but not sorted controls were cultured with allogeneic Tlymphocytes for 5 days then (³H) TdR incorporation determined. Resultsare expressed as the mean of triplicate counts” SEM. Data are from arepresentative experiment of three performed.

FIG. 6 shows CMRF-56 and CD83 reactivity with (A) isolated LC (B)isolated dermal DC and (C) in vitro generated DC. LC and dermal DCpreparations were double labelled with CMRF-56, CMRF-44 and CD83 vsHLA-DR. In vitro generated DC were double labelled with anti-CD1a,CMRF-44 and CMRF-56 vs CD14-PE. In all cases, the gates delineatingpositive staining were set on the basis of negative control staining.The percentages shown on the right of each dot plot indicate thepercentage of either LC, dermal DC or in vitro generated DC thatexpressed the relevant antigen. Data are from representative experimentsof three performed with each type of cell preparation.

FIG. 7 shows CMRF-56 and CD83 reactivity with isolated SF-DC and tonsilDC, before and after in vitro culture. Preparations of (A) Tonsil DC and(B) SF-DC were double labelled with CMRF-56, CD83/FITC SAM vs HLA-DR-PEbefore and after 16 hr culture in medium. In all cases gates delineatingpositive staining were set on the basis of negative control staining.Data are from representative experiments of three performed on each typeof DC preparation.

DESCRIPTION OF THE INVENTION

As indicated above, in a primary aspect the present invention providesimmunological reagents (antibodies) capable of specifically binding toactivated DC. The antibodies bind to a novel activation antigen on DCwhich has been called CMRF-56 antigen.

It will be appreciated that the antibodies which bind activation antigenCMRF-56 can be in the form of antisera containing polyclonal antibodiesor, as is preferred. monoclonal antibodies may be obtained by use ofhybridoma technology. Still further, antibodies or binding fragments canbe produced using biochemical or recombinant DNA techniques.

It is most desirable for the immunological reagents of the invention tobe monoclonal antibodies or binding fragments of such antibodies. Thegeneral procedure of Kohler and Milstein²¹ is therefore used. Generally,this procedure involves obtaining antibody-producing cells from theanimal and fusing the antibody-producing cells with strains of myelomacells to produce hybridomas. These hybridomas are grown or cultured toproduce monoclonal antibodies specific for dendritic cells.

An example of the procedure using myeloma cell line NS-1 is given below.Cell line NS-1 is obtainable from Professor C Milstein, MRC Laboratoryof Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.

Other myeloma cell lines are known in the art and include, for example,the following cell lines: X63Ag8 653, SP2/0, FO and NSO/1. Cell lineswhich neither synthesize nor secrete immunoglobulin heavy or lightchains (eg SP2/0) are generally preferred to cell lines which synthesisebut do not secrete, immunoglobulin chains.

If desired, antibody fragments can be prepared by controlled proteasedigestion of whole immunoglobulin molecules as described in Tjissen²².

Alternatively, antibody fragments can be prepared using molecularbiological techniques by isolating, from hybridoma cells, the geneticmaterial encoding the variable regions of the heavy, light or bothchains of the monoclonal antibodies and expressing them in suitableorganisms for the product of recombinant antigen binding fragments-(Fv,ScFv, Fab etc.) of the monoclonal antibody²³.

By way of illustration of the invention, the generation andcharacterisation of a monoclonal antibody, designated mAb CMRF-56,capable of binding to an epitope on an activation antigen CMRF-56 ofhuman dendritic cells will now be described. From this description,those persons skilled in this art will also appreciate how otherantibodies (or their binding fragments) which bind to activation antigenCMRF-56 can be obtained for use in the extraction of human DC or DC fromother animals.

METHODS

Monoclonal Antibodies and Immunolabelling

The monoclonal antibodies CMRF-15 (erythrocyte sialoglycoprotein, IgM),CMRF31 (CD14, IgG2a), CMRF-44 (IgM) and biotinylated CMRF-44 wereproduced in this laboratory. HB15a (CD83, IgG2b) was a gift from Dr TTedder, Duke University, North Carolina. The CD19 mAb FMC63 (IgG2a) andthe isotype control mAb X63 (IgG 1), Sal4 (IgG2b) and Sal5 (IgG2a) werea gift from Prof H Zola (Flinders Medical Centre, Adelaide, Australia).The CD1a mAb NaI/34 was a gift from Prof A McMichael (Institute ofMolecular Medicine, Oxford, UK). HuNK-2 (CD16, IgG2a) was a gift fromProf I McKenzie (Austin Research Institute, Melbourne, Australia. OKT3(CD3, IgG2a). HNK-1 (CD57, IgM) and OKM1 (CD11b, IgG1) were producedfrom hybridomas obtained from the ATCC. Phycoerythrin conjugatedantibodies to CD3(leu4, IgG1), CD14 (leuM3, IgG2b), CD16(leu11c, IgG1),CD19 (leuM12, IgG1) and HLA-DR (L243, IgG2a) antigens were purchasedfrom Becton Dickinson, Mountain View, Calif. Flourescein isothiocynateconjugated sheep anti-mouse (FITC-SAM) was purchased from Silenus,Hawthorn, Australia. Labelling was carried out on ice using standardtechniques. Briefly, cells were incubated with primary antibody (30min), washed then incubated with FITC-SAM prior to further washing andanalysis. Double labelling of mAb/FITC-SAM labelled cells was carriedout following a further incubation of cells in 10% mouse serum for fiveminutes followed by addition of PE conjugated or biotinylated secondantibody. For biotinylated antibodies a further washing step wasfollowed by incubation (30 min) with avidin-PE (Becton Dickinson). Cellswere analysed or sorted on a FACS Vantage (Becton Dickinson). Samplesthat could not be analysed immediately were fixed in 1% paraformaldehydeand stored at 4° C.

To study capping of the relevent antigens, L428 cells were labelled witheither sal4 or HB15/PE-SAM then incubated at 37° C. for 60 min. Cellswere then washed at 4° C., then labelled (on ice) with eitherCMRF-56-FITC, L243-FITC, X63-FITC or FITC-SAM.

Generation of the CMRF-56 mAb

A balb/c mouse was immunised with the HD-derived cell line L428 and thesplenocytes fused with the myeloma line NS-1 four days later. TheCMRF-56 hybridoma was cloned thrice by limiting dilution and used togenerate ascites fluid. Isotyping was performed using an indirect ELISAkit (Sigma, St.Louis, Mo.). Purified CMRF-56 was prepared utilisingProtein A chromatography, and biotinylated using Biotin-X-NHS(Calbiochem, La Jolla, Calif.). Briefly CMRF-56 at 2 mg/ml in 0.05 MNaHCO₃ (pH 8.5) was incubated with Biotin-X-NHS (7.5 ug/ml, Calbiochem,La Jolla, Calif.) for 30 min (RT) prior to dialysis.

Cell Lines

T cell lines (HSB-2, Molt 4 and Jurkat), EBV transformed B cell lines(WT49, Mann). Burkitt's lymphoma lines (Raji and Daudi), pre-B (Nalm 6),myeloerythroid (K562) and monocytoid leukemia (HL60, U937, KG1, KG1a,THP-1, HEL) leukemia cell lines were grown in medium (10% FCS (IrvineScientific, Santa Anna, Calif.) in RPMI1640 (Gibco, Auckland, NewZealand) supplemented with 2 mM glutamine, 0.06 g/l penicillin and0.1g/l streptomycin). The Hodgkins cell line L428 was obtained from Dr VDiehl (Clinik for Innere Medizine, Cologne, Germany) and the Hodgkinscell lines KM-H2 and HDLM-2 (grown in 20% medium) were obtained from DrH G Drexler (German Collection of Micro-organisms and Cell Cultures,Braunschweig, Germany).

Lymphocyte, Granulocyte and Monocyte Preparation

Blood was obtained from volunteer donors with appropriate informedconsent according to Ethical Committee guidelines. Peripheral bloodmononuclear cells (PBMC) were prepared by centrifugation over sterileFicoll/Hypaque (d=1.077g/cm³, Pharmacia, Uppsala, Sweden) gradients. Tlymphocyte-enriched fractions (ER⁻) and non-T fractions (ER⁻)wereprepared from PBMC by resetting with neuraminidase treated sheeperythrocytes as described previously³⁴. Granulocytes were prepared fromperipheral blood following dextran sedimentation of RBC as describedpreviously³⁴. Activated T lymphocytes were prepared by culture ofER⁺PBMC (2×10⁶/ml) in medium supplemented with either 5 ug/ml PHA(Sigma) or the phobol ester phorbol 12—myristate 13 acetate (PMA, Sigma)at 25 ng/ml plus the calcium ionophore A23187 (Sigma) at 500 ng/ml.

ER⁻PBMC were used as an enriched source of monocytes. Activatedmonocytes were obtained by culture of ER⁻PBMC (2×10⁶/ml) in mediumsupplemented with either IFNγ (500 U/ml, a gift from BoehringerIngelheim, Germany), bacterial LPS (100 ng/ml), TNFα (20 ng/ml) orGM-CSF (500 u/ml, Novartis). Monocyte populations were monitored bydouble labelling with CD14-PE.

The effectivness of the in vitro activation was determined by monitoringby flow cytometry changes in the expression of the activation antigensCD25, CD71, HLA-DR and CMRF-44.

Dendritic Cell Preparation

Highly enriched DC populations were prepared using establishedlaboratory methods:

i) Resting DC were prepared by direct immunodepletion^(33.34). BrieflyER⁻PBMC were labelled with a mix of CD3, CD11b, CD14, CD16 and CD19 mAb.After incubation with MACS magnetic microspheres (Miltenyi Biotech,Germany) labelled cells were removed by magnetic immunodepletion and themAb negative cells were then labelled with FITC-SAM and further purifiedby FACS sorting. In a number of experiments resting DC were thencultured (37° C., 5% CO₂) in medium (2×10⁶/ml) prior to analysis.

ii) Cultured low density blood DC were prepared from cultured (16 h. 37°C., 5% CO₂) ER⁻PBMC³⁶. The low density fraction was then isolated bycentrifgation over a Nycodenz (Nycomed Pharma, Norway) gradient³⁶ andused either directly as a DC enriched (10-30%) fraction or furtherpurified by immunodepletion as described above.

iii) LC and dermal DC were isolated³³ from skin (obtained with consent)separated into epidermal sheets and dermis by overnight digestion (4°C.) with dispase (0.25% in PBS, Boehringer-Mannheim). Epidermal cell(EC) suspensions were produced by disaggregation of the tissue through acell dissociation cup (grade 40 mesh, Sigma) in the presence of 0.25%Typsin (Sigma). Fresh LC were enriched (2-15%) at this stage bylymphoprep gradient as described³⁷. Dermal cell suspensions wereobtained from dermal sheets by incubation (1 h, 37° C.), withcollagenase D (Boehringer-Mannheim, 1 mg/ml) and DNAase I in medium. Asingle cell suspension was obtained by filtering through nylon mesh (80μm) and following centrifugation over a lymphoprep gradient (d=1.077g/cm³, 10 min, 500×g) the low density fraction were utilised as anenriched (30-50%) dermal DC population.

iv) Synovial fluid DC (SFDC) were isolated as previously described³⁸.Following informed consent SF was collected by routine knee jointaspirations from patients with chronic arthritis into EDTA blood tubes.ER⁻ cells were labelled with a mix of mAb against CD3, CD14, CD15, CD16and CD19 and depleted using immunomagnetic MACS beads as described abovefor preparation of blood DC. Residual labelled cells were furtherdepleted using a FACS. The remaining unlabelled MHC class II positivecells constituted the SFDC population.

v) Tonsil DC were prepared from tonsils obtained at routinetonsillectomies, following informed consent. These were processedimmediately and a single cell suspension prepared by mincing the tissuefinely and passing the material through a wire mesh sieve. Mononuclearcells were isolated over a F/H density gradient and Tonsil DC isolatedas described above for SFDC.

vi) In vitro derived DC were generated from the adherent fraction ofPBMC obtained following 2 h culture (37° C.) in Falcon 6 well plates(BD). Adherent cells were cultured in medium supplemented with GM-CSF(800 U/ml) and IL-4 (500 U/ml) for five days, whereupon TNFα was addedto a final concentration of 20 ng/ml and cells cultured for a furthertwo days before analysis.

Immunohistology

Cryostat cut en face sections (7 μm) of tonsil and lymph node (obtainedwith appropriate ethical permission as approved by the Canterbury HealthEthical Committee) were allowed to dry overnight, then fixed for 10 minin ice cold acetone and air dried for 30 min. Sections were incubatedwith 10% goat serum prior to incubation with primary monoclonal antibody(mAb) followed by biotinylated goat anti-mouse Ig (DAKO) and thenaddition of streptavidin-HRP (DAKO). Slides were washed 3 times with TBSbetween each 30 min incubation. Enzymatic activity was revealed with3.3′-diaminobenzidine solution. After a final wash in PBS slides werecounterstained (standard H&E stain) then mounted.

Immunofluorescent double labelling of acetone fixed tissue sections wascarried out as described above for cell suspensions.

Functional Assays

Allogeneic MLR: 10⁵ T lymphocytes were cultured at 37° C. in 5% CO₂ in96 well plates with triplicate graduated numbers of sorted APC subsetsobtained from a single allogeneic donor. Wells were pulsed for 12 hourswith 0.5 Ci tritiated thymidine (Amersham) immediately prior to harvestat five days. Cells were harvested onto filter paper and thymidineincorporation was measured with a liquid scintillation counter. Data areexpressed as mean CPM of triplicate wells ±SD. Control wells containingT cells or APC alone incorporated <500 cpm of tritiated thymidine in allexperiments.

Preparation of CD83 Transfectants

COS-7 cells grown in medium were plated on Nunc petri dishes toapproximately 50% confluence. Transfection was carried out byelectroporation (300 V. 500 uF) of cells (4×10⁶ in 400 ul medium) with 2ug of CD83 plasmid (CDM8—CD83 kindly provided by Dr Tedder) or controlplasmid in a biorad Gene Pulser. Cells were then cultured in medium 72 hprior to immunofluorescent analysis with the HB15a mAb to confirm CD83expression.

Expression of CD83 Fusion Proteins

CD83-Ig was expressed in eukaryotic cells. A DNA fragment of CD83extracellular domain (including signal peptide) was amplified from aCD83 cDNA by polymerase chain reaction (PCR) usinga pair of primers(MK001:5′-CCCAAG CTT ATG TCG CGC GGC CTC CAG-3′ (forward) (SEQ ID NO:1)and MK002:5′-GCG AAT TCA CTT ACC TGT CTC CGC TCT GTA TTT CTT-3′(reverse) (SEQ ID NO:2) with unique HindIII and EcoRI sites underlined).The resultant fragment was digested with EcoRI and HindIII, and ligatedto EcoRI- and HindIII-digested pBluescript to generate pBS-CD83 for DNAsequencing. After confirming the DNA sequence, the fragment was excisedwith EcoRI and HindIII, and ligated to EcoRI- and HindIII-digested pIGvector. The vector was transfected to COS cells by electroporation, andCD83-Ig was purified from the conditioned media of the COS cells usingprotein A column chromatography. CD83-Histamine (CD83-Hist) wasexpressed in prokaryotic cells. A DNA fragment of CD83 extracellulardomain (excluding signal peptide) was amplified from a CD83 cDNA by PCRusing a pair of primers (MK010:5′-GAA GAT CTA CGC CGG AGG TGA AGG TG-3′(forward) (SEQ ID NO:3) and MK011:5′-GAA GAT CTC TCC GCT CTG TAT TTCTT-3′ (reverse) with an unique Bg1 II site (underlined). The resultantfragment was digested with Bg1 II, and ligated to Bg1 II-digested pQE12to generate pQE-CD83. After confirms the DNA sequence and inframestatus, the vector was used to transform XL-1 blue bacteria andCD83-Hist fusion protein was induced by adding IPTG to the bacteriaculture. The fusion protein was purified from the bacteria lysate usingNi-NTA resin column chromatography.

CD83 ELISA

Binding of CMRF-56 and HB15 to CD83 constructs was analysed by ELISA.ELISA plates (Maxisorp, Nunc) were coated by incubation (37° C. 1 hr)with CD83-Ig, CD83-H or human Ig (hIg, salt precipitated) at aconcentration of 10 ug/ml. Following blocking (2% BSA/PBS) wells wereincubated (1 h, 37° C.) with either culture supernatant, ascites orpurified mAb diluted in 1% BSA/PBS. Following washing (0.1% Tween20/PBS) plates were incubated (1 hr, 37° C.) with GAM-HRP (Dako, 1:1500)prior to washing and colour development using o-phenylenediamine (OPD)substrate. Plates were then analysed (492 nm/650 nm) on a MRX microplatereader (Dynatech Laboratories).

Enzyme and Inhibition Studies

The enzyme susceptibility of the CMRF-56 antigen was tested byincubating (30 min, 37° C.) the cell line L428 in PBS containing eitherpronase (50 ug/ml, Sigma) or neuraminidase (0.1 U/ml, Behring, Marburg,Germany). Cells were washed (×3) prior to analysis by flow cytometry.The enzyme induced changes in the strength of mAb binding weredetermined by comparison of the MFI of treated cells with that of cellsincubated in PBS alone.

N-linked glycosylation of glycoproteins was blocked by incubation (12 h.37° C.) in medium containing either 0 or 10 ug/ml of tunicamycin(Sigma). The effect of treatment on mAb binding was determined by flowcytometry.

Immunoprecipitation

Cells were labelled using three methods (i) cell surface labelling withBiotin-X-NHS (Calbiochem)^(20.39), (ii) cell surface sialic acidlabelling with biotin hydrazide (Calbiochem)⁴⁰ or (iii) biosyntheticallylabelled with ³⁵S (NEN, Boston. Mass.)²⁰. Following labelling cells weresolubilised by incubation (1 hr on ice) of cells (4×10⁷) in 1 ml lysisbuffer (100 mM Tris, 150 mM NaCl, 0.02% NaN3, pH 7.8) containing either0.5% Triton X-100 or 0.25% CHAPS and supplemented with enzyme inhibitorComplete™ (Boehringer). Following centrifugation (10000 g, 10 min),solubilized proteins were analysed by either (i) immunoprecipitationusing rabbit anti-mouse immunoglobulin covalently coupled toCNBr-activated Sepharose 4B (RAM-Sepharose) as describedpreviously^(20.39) or (ii) immunoabsorption of antigen by mAb capturedon Maxsorp ELISA plates⁴¹. Eluted protein was analysed by gradientSDS-PAGE in combination with either autoradiography or Western blottingin combination with chemiluminescent visualisation.

Lipid extracts were prepared from L428 as described previously²⁰. Slotblotting of whole cell lysates (prepared as described above) or lipidextracted material and subsequent immunostaining was carried out asdescribed previously²⁰.

RESULTS

Generation of CMRF-56 mAb

Hybridomas were generated by fusion of NS-1 mycloma cells with spleencells obtained from a mouse immunised with the HD-derived cell lineL428. Hybridomas producing mAb reactive with L428 but not PBMC wereidentified, then analysed for reactivity with cultured low density DC.The mAb CMRF-56 (IgG₁) labelled a cell population within these DCpreparations and was characterised as described below.

CMRF-56 Reactivity with Normal Haemopotetic Non-DC Populations

Cell surface expression of the CMRF-56 antigen on isolated blood andtonsil leucocyte populations was analysed by both single and doublelabelling in conjunction with flow cytometry. The CMRF-56 mAb did notreact with circulating PBMC (n=5), peripheral blood granulocytes (n=3,FIG. 1A), the CD3⁺ population within ER⁻PBMC preparations (n=4, FIG. 1A)or the CD16⁺, CD14⁺ and CD19⁺ populations within ER⁻PBMC preparations(n=6) (FIG. 1A).

In vitro culture (16 h, 37° C.) of ER⁺PBMC (n=3) for 24 and 72 h inmedium or medium supplemented with PHA or PMA+Cal (n=3) did not induceCMRF-56 antigen expression on the CD3⁺ population. Culture of ER⁻PBMC inmedium (16 h, 37° C.) induced the expression of the CMRF-56, CD83 andCMRF-44 antigens on a subpopulation of the CD19⁺ population whereas theCD19⁻ population (including CD14⁻ monocytes) lacked these antigens (FIG.1B).The culture of ER⁻ and ER⁺ preparations in the presence of PMA/Calinduced CMRF-56 and CD83 expression on the CD19⁺ cells present. Cultureof ER⁻ PBMC for 24 h and 72 h in medium supplemented with additionalLPS, IFNγ, GM-CSF or TNFα failed to induce the expression of CMRF-56 onthe CD14⁺ monocyte population despite the induction of changes inCMRF-44 or MHC class II antigen expression (data not shown, n=3).Analysis by flow cytometry of isolated tonsillar lymphocytes confirmedthat CMRF-56 did not label with tonsil T lymphocytes (n=4) but, incommon with CD83 and CMRF-44, did label a proportion of the tonsil Blymphocytes with moderate intensity (FIG. 1C).

In all tonsil lymphocyte preparations analysed (n=5) there was a cleardifference in the percentage of B lymphocytes labelling with the mAb:the CMRF-44 antigen was expressed on a higher percentage and the CD83antigen on a lower percentage of cells than the CMRF-56 antigen.

Reactivity with Cell Lines and Transfectants

The cell surface expression of CMRF-56 on human cell lines was analysedby flow cytometry. The CMRF-56 antigen was expressed at detectablelevels on a number of B cell lines (Mann, RaJi,) and HD-derived celllines (L428, KM-H2, HDLM-2) with the strongest staining noted on theL428 (FIG. 2A) and Mann cell lines. Cell lines that did not expressdetectable levels of CMRF-56 antigen included the myelo-erythroid K562line, the T lymphoid lines HSB2 and Molt 4, the myeloid monocytoid celllines NB4, THP1, U937, KG1 and KG1a and the pre B lymphoid line NALM6.The CMRF-56 mAb did not react with the CD83⁺ T lymphoid cell line Jurkat(FIG. 2A) and as shown in FIG. 2B did not label CD83 positive COS celltransfectants (n=3).

The CMRF-56 antigen showed significant capping on a proportion of L428cells by CMRF-56 mAb FITC-SAM. The CD83 antigen was also capped byHB15/PE-SAM into discrete patches on 60% of stained cells. Of the cellscapped with CMRF-56 and subsequently stained with CD83, a proportionshowed residual evenly distributed CD83 staining of the membraneindicating independent membrane molecular localisation of the twoantigens.

Biochemical Analysis

The sensitivity of CMRF-56 antigen to enzyme digestion or blockage ofn-linked glycosylation was examined by flow cytometry. Increased bindingof the CMRF-56 mAb to L428 cells was observed following treatment of thecells with either neuramnidase (1.5 fold increase, sd=0.2, n=5) orpronase (1.4 fold increase, sd=0.25, n=4). Preincubation withtunicamycin did not significantly alter observed binding. Immunostainingof L428 detergent lysates applied to NC membranes demonstrated that theCMRF-56 antigen was effectively solubilised by the non-ionic detergentTRITON X-100 and the zwitteronic detergent CHAPS. Numerousimmunoprecipitation experiments from lysates prepared following L428cell surface protein (biotin), cell surface sialic acid (biotinhydrazide) or metabolic (³⁵S) labelling failed to identify the CMRF-56antigen despite co-precipitation of appropriate molecular weightproducts with the anti-MHC class II and CD83 reagents (data not shown).Western blotting of L428 and Mann cell line lysates similarly failed toidentify the CMRF-56 antigen. Immunostaining of L428 lipid and non-lipidextracts applied to nitrocellulose membranes did not detect CMRF-56antigen in either fraction, suggesting that the CMRF-56 antigen epitopeis sensitive to organic solvents (data not shown). The reactivity ofCMRF-56 with both CD83-Ig and CD83-hist constructs was analysed by ELISA(FIG. 3). In contrast to the CD83 mAb, CMRF-56 did not bind to eitherthe CD83 Ig (n=3) or the CD83-hist recombinant material (n=3).

CMRF-56 Reactivity with Isolated DC

The reactivity of CMRF-56 with isolated DC populations was examined byindirect immunofluorescence and flow cytometry.

Directly isolated fresh DC (FIG. 5A) did not express detectable levelsof either the CMRF-56 or CD83 antigens. However expression of bothantigens was rapidly induced on directly isolated DC within 6 hrs of invitro culture. In contrast, expression of the CMRF-44 antigen wasconsistently detected on a subpopulation of directly isolated DC andfurther upregulation of the CMRF-44 antigen preceded that of both theCMRF-56 and CD83 antigens.

Analysis of the DC enriched low density fraction of cultured ER⁻PBMCinvariably identified a subpopulation of CMRF-56⁺ cells (10-30%, n=20)identical to the DC populations detected by CD83 and CMRF-44 (FIG. 4A).Double labelling (FIG. 4B) confirmed that the CMRF-56 reactivity wasassociated with the lin⁻, CMRF-44⁺ and CD83⁺ DC populations. FACSsorting of low density ER⁻PBMC on the basis of CMRF-56 expressionclearly demonstrated that potent allostimulatory activity was associatedwith the CMRF-56⁺ population and that the CMRF-56⁻ population was onlyweakly stimulatory (n=3, FIG. 4C). Binding of the mAb did not affect theDC allostimulatory activity.

Flow cytometric analysis of isolated LC (n=3)demonstrated thatapproximately 40% of these cells express the CMRF-56 and CMRF-44antigens at high density, with CD83 being expressed weakly on asignificantly lower percentage of cells (FIG. 6A). Dermal DC (n=3),although strongly CMRF-44 and CMRF-56 positive, showed only weakstaining of a subpopulation of cells with CD83 (FIG. 6B).

Directly isolated SFDC, although lacking the CD83 antigen, contained asubpopulation of CMRF-56⁺ cells (n=5, FIG. 7A). Following in vitroculture of these SF DC preparations further upregulation of both theCMRF-56 and CD83 antigens was observed.

Tonsil DC prepared by direct immunodepletion were, in common withfreshly isolated blood DC, CD83 and CMRF-56 negative but expressed bothantigens in high density after a period of in vitro culture (n=5, FIG.7B).

In vitro generated Mo-DC populations were also studied (n=3). Followingculture of monocytes in the presence of GM-CSF. L-4 and TNFα theresulting Mo-.DC were strongly CMRF-56⁺. The percentage of CMRF-56⁺cells was significantly less than the percentage of CD1a⁺ positive cells(FIG. 6C).

Immunohistological Analysis of CMRF-56 Expression

Immunohistological staining of lymph node and tonsil sections detectedweak CMRF-56 antigen expression on the germinal centre lymphocytes. andstrong expression on scattered interfollicular (T zone) cells.Immunofluorescent double labelling of tonsil sections demonstrated thatthe CMRF-56 positive interfollicular cells lacked CD 19 and CD20 butexpressed CD86. Double labelling with CMRF-56 and CD83 demonstrated thatwithin the interfollicular regions CMRF-56 antigen was expressed on alower number of cells than CD83 and that a subpopulation of the CMRF-56positive population did not express CD83.

DISCUSSION

Characterisation of the mAb CMRF-56 has established that it recognises apreviously undefined antigenic epitope with restricted expression onhuman DC populations. Circulating blood leucocytes did not express theCMRF-56 antigen and following either culture alone or in vitroactivation. CMRF-56 antigen expression was detected only within the lowdensity (DC enriched) fraction of cultured PBMC and on a subpopulationof CD19⁺ lymphocytes. Immunolabelling and FACS sorting of the lowdensity fraction of cultured PBMC confirmed that CMRF-56 was stainingthe DC population within these preparations. The finding thatcirculating blood DC are CMRF56 but express the antigen in high densitywithin 6 h culture confirmed that CMRF-56 recognises a earlydifferentiation/activation marker on DC. The CMRF-56 antigen was alsoexpressed on other DC populations including isolated LC and dermal DC.Rapid upregulation of the CMRF-56 antigen on tonsil and Synovial fluidDC occurred after a short period of in vitro culture. The CMRF-56antigen can be clearly distinguished from the CMRF-44 on the basis ofits absense from CMRF-44⁺ cells eg. in vitro cultured and IFNγ activatedmonocytes as well as freshly isolated blood DC. Likewise, the lack ofCMRF-56 reactivity with CD83 transfectants, CD83 recombinant proteinsand the CD83⁺ cell line Jurkat clearly distinguished these two antigens.At present the only selective DC surface markers available areCMRF-44^(13.20) and CD83^(18.19), which also recognise early activationmarkers on DC. The CMRF-44 antigen, but not the CD83 and CMRF-56antigens, is expressed on a subpopulation of circulating DC³¹. Althoughall three markers are rapidly upregulated on DC with culture, as shownin this study upregulation of CMRF-44 on isolated blood DC, clearlyprecedes that of CMRF-56 and CD83 antigens. Analysis of isolated dermalDC, LC, synovial fluid DC and tonsil DC suggests that expression of theCMRF-56 antigen precedes CD83 expression on these populations.

Thus it appears that these three distinct DC differentiation/activationantigens upregulate in the order of CMRF-44 antigen, CMRF-56 antigen andthen the CD83 antigen. However, this interpretation may be influenced bythe fact that the expression of the CMRF-44 and CMRF-56 antigens onisolated DC is maintained throughout a short period of in vitro culture,whereas in some experiments the surface CD83 antigen labelling decreasesafter 24 h. This downregulation of CD83 antigen expression may be due tothe cleavage of surface protein and release of soluble CD83 which hasbeen reported to occur with activated B lymphocytes³⁶.

CMRF-56 and CD83 differed considerably in terms of their reactivity withIDC in tonsil sections. Double labelling demonstrated that the CMRF-56antigen was expressed. on a considerably lower number of cells than CD83in the interfollicular zone and that populations of both CMRF-56⁺/CD83⁺,CMRF-56⁻/CD83⁺ and CMR-56⁺, CD83⁻ cells were present. Previous studieshave demonstrated that CD83 is expressed by a subset of IDC withintonsil tissue. The expression of the CMRF-56 antigen on a CD19⁻,CD20⁻,HLA-DR⁺ population within the interfollicular zone that includes apopulation of CD83 cells provides further evidence that the CMRF-56 andCD83 antigens are expressed at different stages of DCdifferentiation/activation. However, the absence of CMRF-56 antigen on asubpopulation of CD83⁺ IDC contrasts with the results obtained usingisolated tonsil DC populations. Thus CD83⁺. CMRF-56⁻ DC populations werenot detected in any of the preparations analyzed either before or afterin vitro culture. This may in the case of isolated tonsil DC reflect thedifficulty in isolating all the cell populations from the tissueparticularly without exposing the isolated cells to in vitro enzymedigestion. Interestingly, although the majority of germinal centre Bcells expressed low density CMRF-56 and CD83 antigens when analysed insitu, only a subpopulation of isolated tonsillar B cells expressed theseantigens, suggesting that the release of B cells from the tissue matrixmay be unrepresentative.

CMRF-56 expression on human cell lines parallels that of the CMRF-44 andCD83 antigens, in many respects being restricted to HD derived and Bcell lines whereas cell lines of myeloid origin lack these antigens. Asimilar pattern is observed following activation of ER⁻PBMC or ER⁺PBMCpopulations, with expression of CMRF56 and CD83 being readily inducibleon B lymphocytes. Although all blood B lymphocytes express theseantigens following in vitro activation, these antigens have distinctlydifferent levels of expression on isolated tonsil lymphocytes, CD83being expressed on a considerably lower percentage of B lymphocytes thanCMRF-56, whilst CMRF-44 had considerably higher expression.

Negligible expression of these antigens on the CD14⁻ monocyte populationwas observed using a range of single stimuli. Nonetheless, CD83expression and CMRF-44 can be induced on cells of myeloid originfollowing long term culture in the presence of particular cytoidnecombinations. These cells closely resemble DC in terms of function andphenotype and as shown in this study In vitro these Mo-DC also expressthe CMRF-56 antigen.

It is clear that the specificity of CMRF-56 and CD83 for DC populationsis not absolute, but the study of these antigens in conjunction with Blymphocyte markers provides a highly selective means of identifying DCpopulations at an early stage of activation, both in situ and withinisolated leucocyte populations. The upregulation of these molecules isassociated with a phase of significant activation of DC function. Thus,these DC upregulate the costimulator molecules CD80,CD86^(30.32.19.14.43), CD40³³ and adhesion molecules such asICAM-1^(7.44).

In summary, the CMRF-56 antigen is as are the other associated antigensCMRF-44 and CD83 expressed on the L428 cell line. Nonetheless, theserological data provided clearly distinguishes the CMRF-56 antigenicepitope from the CMRF-44 antigen. The CD83 antigen which at aserological level has some parallels with the CMRF-56 antigen is clearlydistinguished from it as a cell surface protein that caps independentlyof CMRF-56 antigen. Further evidence was obtained for the distinctnature of these two antigens by demonstrating that mAb CMRF-56 did notbind CD83transfected cells or recombinant material. Thus mAb CMRF-56becomes a further mAb with specificity for DC.

The CMRF-56 mAb does not label cytoline or LPS stimulated bloodmonocytes over the 48 hr period of observation. This makes CMRF-56 mAbperhaps particularly useful as a reagent for determining committedmonocytic cells from committed DC precursors. It is clear that theCMRF-56 antigen upregulates as part of the DC activation/differentiationpathway. The kinetics of upregulation documented in FIG. 5 suggest theCMRF-56 antigen is expressed later than CD83 but persists larger thanCD83 which appears to down regulate after 48 hrs.

INDUSTRIAL APPLICATION

There are a number of uses to which the antibodies of the invention(which recognise and bind to the activation antigen CMRF-56) can be put.Such uses include (1) the identification (for diagnostic purposes) ofactivated DC; and (2) the purification/concentration of activated DC,and these uses accordingly represent further aspects of this invention.

Diagnostic applications of the present exemplary mAb CMRF-56 includeallowing for assessment of activated (CMRF-56 positive) againstnon-activated (CMRF-56 negative) DC, which may be of use in thediagnosis and/or therapy of diseases such as cancer.

In such applications, any immunological-based assay procedures known inthe art could be employed for quantifying the amount of activated DC ina sample. Such procedures are summarised in TiJssen²⁴ such as flowcytometry. ELISA, RIA and fluorescence microscopy among others.

In terms of isolation of activated DC, once again any process orpurification system which employs the antibodies (or their bindingfragments) as the primary immunological reagent can be used. Many suchprocesses are known, as are purification systems which allow for theseprocesses to be put into effect. An example of a commercially availablepurification system is the avidin-biotin immunoaffinity system²⁹ fromCellPro, Inc., Washington, USA. See also U.S. Pat. Nos. 5,215.927,5,225,353, 5,262,334, 5,240,856 and PCT/US91/07646 published Apr. 30,1992, all incorporated herein by reference. This system employs directlyor indirectly a biotinylated monoclonal antibody directed against atarget cell and a column containing immunobilized avidin and can bereadily adapted to extract activated human dendritic cells, in this casefrom human peripheral blood, using the exemplary mAb CMRF-56 as follows:

1. A sample of human peripheral blood containing the human dendriticcells is mixed with biotinylated mAb CMRF-56 and incubated to allowformation of mAb CMRF-56/human DC complexes.

2. Following incubation, the mixture is introduced into a CellProcontinuous-flow immunoadsorption column filled with avidin-coated beads,the strong affinity between biotin and avidin causing the biotin-coatedmAb CMRF-56 (together with the human DC to which they have bound) toadhere to the avidin-coated beads.

3. After unwanted cells present in the mixture are washed away, capturedactivated human DC are removed from the column by gentle agitation andare available for use.

Variations on this theme using mAb CMRF-56 as primary antibody (to bindto activated DC) and a biotinylated secondary antibody (to bind to mAbCMRF-56) can also be employed.

It will be appreciated that before admixture with mAb CMRF-56 inaccordance with the above protocol, the human peripheral blood sampleshould be treated to ensure that the DC the sample contains areactivated. This can easily be achieved by, for example, overnightincubation of the sample.

For use in the above protocol, mAb CMRF-56 can be biotinylated by anyone of a number of conventional methods. For example, the biotinylationprocedure of Berenson et al²⁹ can be employed.

A possible and preferred preliminary step in the methods outlined aboveis the enrichment of DC in the sample by gradient centrifugation²⁵⁻²⁷.While this optional enrichment step can employ any suitable knowngradient medium (such as albumin or metrizamide), it is howeverpreferred that a Nycodenz medium (Nycomed Pharma, Oslo, Norway) beused²⁸ in relation to 16 hour cultured T lymphocyte-depleted peripheralblood mononuclear cells. The applicants have found that use of thisgradient reliably yields a population of low density cells that ishighly enriched for DC.

It will be apparent to one skilled in the art that there are numerousother means of immunoselection of dendritic cells, in addition toavidin-biotin immunoaffinity chromatography. These include, but are notlimited to, immunoselection using magnetic beads, ferrofluids,dipsticks, petri dishes, and a wide variety of other solid phases thatcan be derivatized so as to specifically bind mAb CMRF-56 labelled DC.

Once purified/concentrated by the above or any other suitable process,the activated DC can be employed in research or in commercialapplications. One such potentially commercial application for activatedDC is as part of an immunopotentiating composition together with anantigen protective against disease, for either prophylaxis or therapy.It is believed that such compositions would increase both the speed andefficiency of the immune response generated against the protectiveantigen.

Other applications of the activated DC will of course be apparent tothose persons skilled in this art.

Another contemplated application of the mAb CMRF-56 is in targetingactivated DC in patients to induce immunosuppression.

It will be understood that the above description is exemplary only andthat the present invention is not limited thereto.

DEPOSIT

Hybridoma CMRF-56 (produced using myeloma cell line NS-1) has beendeposited to provide supplemental disclosure of the invention.Deposition was with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, USA as of Oct. 9, 1996under the terms of the Budapest Treaty. All restrictions upon publicaccess to this deposit will be irrevocably removed upon the grant of thepatent and the deposit will be replaced if viable samples cannot bedispensed by the Depository. Hybridoma CMRF-56 has been given ATCCAccession No. 12202.

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4 1 27 DNA Artificial Sequence Description of Artificial SequenceMade inlab 1 cccaagctta tgtcgcgcgg cctccag 27 2 36 DNA Artificial SequenceDescription of Artificial SequenceMade in lab 2 gcgaattcac ttacctgtctccgctctgta tttctt 36 3 26 DNA Artificial Sequence Description ofArtificial SequenceMade in lab 3 gaagatctac gccggaggtg aaggtg 26 4 26DNA Artificial Sequence Description of Artificial SequenceMade in lab 4gaagatctct ccgctctgta tttctt 26

What is claimed is:
 1. An isolated monoclonal antibody CMRF-56 producedby hybridoma ATCC HB 12202 that binds activated dendritic cells (DC). 2.The isolated antibody of claim 1 wherein the antibody binds to anantigen on activated DC which antigen binds to monoclonal antibodyCMRF-56 produced by hybridoma ATCC HB
 12202. 3. A hybridoma cell linehaving Accession No. ATCC HB
 12202. 4. A process for purifying activateddendritic cells (DC) from a sample containing said activated DC, saidprocess comprising the steps of contacting said sample with the antibodyof claim 1 and then recovering activated DC which have bound to saidantibody.
 5. A process for identifying activated dendritic cells (DC) ina sample comprising the steps of contacting said sample with theantibody of claim 1 to form an antibody/activated DC complex; anddetecting the presence of said antibody/DC complex to identify theactivated dendritic cells (DC).
 6. The process of claim 4 wherein theCMRF-56 antibody is biotinylated.
 7. The process of claim 5 wherein theCMRF-56 antibody is biotinylated.